High Osmolarity Glycerol Pathway Research Articles

Shared and redundant proteins coordinate signal cross-talk between MAPK pathways in yeast.

All cells must detect, interpret, and adapt to multiple and concurrent stimuli. While signaling pathways are highly specialized, different pathways often share components or have components with overlapping functions. In the yeast Saccharomyces cerevisiae, the high osmolarity glycerol (HOG) pathway has two seemingly redundant branches, mediated by Sln1 and Sho1. Both branches are activated by osmotic pressure, leading to phosphorylation of the MAPKs Hog1 and Kss1. The mating pathway is activated by pheromone, leading to phosphorylation of the MAPKs Fus3 and Kss1. Given that Kss1 is shared by the two pathways, we investigated its role in signal coordination. We activated both pathways with a combination of salt and pheromone, in cells lacking the shared MAPK and in cells lacking either of the redundant branches of the HOG pathway. By systematically evaluating MAPK activation, translocation, and transcription programs, we determined that Sho1 mediates cross talk between the HOG and mating pathways and does so through Kss1. Further, we show that Kss1 initiates a transcriptional program that is distinct from that induced by Hog1 and Fus3. Our findings reveal how redundant and shared components coordinate concurrent signals and thereby adapt to sudden environmental changes.

The N-terminus of the Aspergillus fumigatus group III hybrid histidine kinase TcsC is essential for its physiological activity and targets the protein to the nucleus.

Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.

Aspergillus nidulans gfdB, Encoding the Hyperosmotic Stress Protein Glycerol-3-phosphate Dehydrogenase, Disrupts Osmoadaptation in Aspergillus wentii.

The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA, but not the gfdB, glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and ΔgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed a canonic hyperosmotic stress response characterized by the upregulation of the trehalose and glycerol metabolism genes (including gfdB), as well as the genes of the high-osmolarity glycerol (HOG) map kinase pathway. The deletion of gfdB caused only negligible alterations in the transcriptome, suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than did A. nidulans, e.g., the bulk upregulation of the glycerol and trehalose metabolism genes, along with the HOG pathway genes, was not detected. The expression of An-gfdB in A. wentii did not abolish osmophily, but it reduced growth and caused much bigger alterations in the transcriptome than did the missing gfdB gene in A. nidulans. Flexible glycerol metabolism and hence, two differently regulated gfd genes, may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

ARV1 deficiency induces lipid bilayer stress and enhances rDNA stability by activating the unfolded protein response in Saccharomyces cerevisiae

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan (RLS) of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the RLS of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner.

Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of S. sclerotiorum (Shk1) revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized SsHog1, which is the downstream kinase of Shk1. To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure SsHog1 and Shk1 knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the SsHog1 knockout mutants demonstrated reduced virulence compared to Shk1 knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and SsHog1 is involved in osmotic adaptation, fungicide sensitivity, and virulence in S. sclerotiorum.

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal

BackgroundAspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant.ResultsIn this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress.ConclusionsOur data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Crucial Roles of the High-Osmolarity Glycerol Pathway in the Antifungal Activity of Isothiocyanates against Cochliobolus heterostrophus.

Isothiocyanates (ITCs) that are found in Brassicaceae exhibited obvious antifungal activity against Cochliobolus heterostrophus, which is the causal agent of southern corn leaf blight. However, the underlying antifungal mechanism of allyl-ITCs (A-ITCs) against C. heterostrophus remains largely unknown. Here, we used transcriptomic analysis to find that the high osmolarity pathway was upregulated significantly when treated with A-ITCs. To investigate the roles of the high osmolarity pathway in adaption to A-ITCs, we constructed Δssk2, Δpbs2, and Δhog1 mutant strains. Deletion of three genes (ChSSK2, ChPBS2, and ChHOG1) involved in the high osmolarity pathway resulted in significantly increased sensitivity of C. heterostrophus to ITCs. In addition, the phosphorylation level of ChHog1 was induced by A-ITC and was dependent on the presence of ChSsk2 and ChPbs2. Moreover, Δssk2, Δpbs2, and Δhog1 mutants exhibited a dramatically decreased virulence on maize leaves. Our findings demonstrated that the high osmolarity pathway played a positive role in ITC tolerance and virulence, which may provide novel insights into developing ITCs as a new fungicide against C. heterostrophus.

The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense.

Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay's controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.

Loss of function of Hog1 improves glycerol assimilation in Saccharomyces cerevisiae

We previously isolated a mutant of Saccharomyces cerevisiae strain 85_9 whose glycerol assimilation was improved through adaptive laboratory evolution. To investigate the mechanism for this improved glycerol assimilation, genome resequencing of the 85_9 strain was performed, and the mutations in the open reading frame of HOG1, SIR3, SSB2, and KGD2 genes were found. Among these, a frameshift mutation in the HOG1 open reading frame was responsible for the improved glycerol assimilation ability of the 85_9 strain. Moreover, the HOG1 gene disruption improved glycerol assimilation. As HOG1 encodes a mitogen-activated protein kinase (MAPK), which is responsible for the signal transduction cascade in response to osmotic stress, namely the high osmolarity glycerol (HOG) pathway, we investigated the effect of the disruption of PBS2 gene encoding MAPK kinase for Hog1 MAPK on glycerol assimilation, revealing that PBS2 disruption can increase glycerol assimilation. These results indicate that loss of function of Hog1 improves glycerol assimilation in S. cerevisiae. However, single disruption of the SSK2, SSK22 and STE11 genes encoding protein kinases responsible for Pbs2 phosphorylation in the HOG pathway did not increase glycerol assimilation, while their triple disruption partially improved glycerol assimilation in S. cerevisiae. In addition, the HOG1 frameshift mutation did not improve glycerol assimilation in the STL1-overexpressing RIM15 disruptant strain, which was previously constructed with high glycerol assimilation ability. Furthermore, the effectiveness of the HOG1 disruptant as a bioproduction host was validated, indicating that the HOG1 CYB2 double disruptant can produce L-lactic acid from glycerol.

Heat shock transcriptional factor HSF1 is activated by phosphorylation in response to ginger oleoresin stress in S. cerevisiae

Ginger in traditional Chinese medicine is widely applied to dispel cold due to ginger's thermogenic effects when consumed. Heat shock response is a classical and highly conserved signaling pathway in cells regulating gene expressions in response to heat stress. However, the potential molecular links between ginger and heat shock response are still unexplored. We described that the ginger oleoresin stress and heat stress produced similar yeast phenotypes and shared genes in RNA-seq analyses. In saccharomyces cerevisiae, a high-osmolarity glycerol pathway (HOG) and critical transcription factors, Msn2/4 and Hsf1 are mainly involved in response to heat stress. Compared with heat shock, we found that only transcription factor Hsf1 was phosphorylated and activated in response to ginger oleoresin stress, accumulating in the nucleus and increasing expression of heat shock proteins such as Hsp104, Hsp78, and Hsp82. Three serine or threonine residues of Hsf1, T162, T163, and S168 were phosphorylated in response to ginger oleoresin stress. These results address the link between ginger and heat shock and their potential molecular mechanism.

The AwHog1 Transcription Factor Influences the Osmotic Stress Response, Mycelium Growth, OTA Production, and Pathogenicity in Aspergillus westerdijkiae fc-1.

Aspergillus westerdijkiae, known as the major ochratoxin A (OTA) producer, usually occurs on agricultural crops, fruits, and dry-cured meats. Microorganisms produce OTA to adapt to the high osmotic pressure environment that is generated during food processing and storage. To investigate the relationship between OTA biosynthesis and the high osmolarity glycerol (HOG) pathway, the transcription factor AwHog1 gene in A. westerdijkiae was functionally characterised by means of a loss-of-function mutant. Our findings demonstrated that the growth and OTA production of a mutant lacking AwHog1 decreased significantly and was more sensitive to high osmotic media. The ΔAwHog1 mutant displayed a lower growth rate and a 73.16% reduction in OTA production in the wheat medium compared to the wild type. After three days of culture, the growth rate of the ΔAwHog1 mutant in medium with 60 g/L NaCl and 150 g/L glucose was slowed down 19.57% and 13.21%, respectively. Additionally, the expression of OTA biosynthesis genes was significantly reduced by the deletion of the AwHog1 gene. The infection ability of the ΔAwHog1 mutant was decreased, and the scab diameter of the pear was 6% smaller than that of the wild type. These data revealed that transcription factor AwHog1 plays a key role in the osmotic response, growth, OTA production, and pathogenicity in A. westerdijkiae.

Investigation of osmotic shock effect on pulsed electric field treated S. cerevisiae yeast cells

Pulsed electric field (PEF) treatment is known to cause plasma membrane permeabilization of microorganisms, an effect known as electroporation. PEF treatment is very attractive since it can achieve permeabilization with or without lethal damage in accordance with desired results. This study aimed to expand the accomplishment of electroporation outcomes by applying sudden post-PEF osmotic composition change of the media. Changes in yeast cells’ viability, size and plasma membrane regeneration rate were evaluated. However, we still have questions about the intracellular biochemical processes responsible for plasma membrane recovery after electroporation. Our suggested candidate is the high osmolarity glycerol (HOG) kinase pathway. The HOG pathway in Saccharomyces cerevisiae yeasts is responsible for volume recovery after dangerous shape modifications and intracellular water disbalance caused by environmental osmotic pressure changes. Thus, we evaluated the HOG pathway inactivation effect on S. cerevisiae’s reaction to PEF treatment. Results showed that Hog1 deficient S. cerevisiae cells were considerably more sensitive to electric field treatment, confirming a link between the HOG pathway and S. cerevisiae recovery process after electroporation. By suddenly changing the osmolarity of the media after PEF we influenced the cells’ plasma membrane recovery rate, severity of permeabilization and survivability of yeast cells. Studies of electroporation in combination with various treatments might improve electric field application range, efficiency, and optimization of the process.

MAP Kinase FgHog1 and Importin β FgNmd5 Regulate Calcium Homeostasis in Fusarium graminearum.

Maintaining cellular calcium (Ca2+) homeostasis is essential for many aspects of cellular life. The high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway responsible for signal integration and transduction plays crucial roles in environmental adaptation, especially in the response to osmotic stress. Hog1 is activated by transient Ca2+ increase in yeast, but the functions of the HOG pathway in Ca2+ homeostasis are largely unknown. We found that the HOG pathway was involved in the regulation of Ca2+ homeostasis in Fusarium graminearum, a devastating fungal pathogen of cereal crops. The deletion mutants of HOG pathway displayed increased sensitivity to Ca2+ and FK506, and elevated intracellular Ca2+ content. Ca2+ treatment induced the phosphorylation of FgHog1, and the phosphorylated FgHog1 was transported into the nucleus by importin β FgNmd5. Moreover, the increased phosphorylation and nuclear accumulation of FgHog1 upon Ca2+ treatment is independent of the calcineurin pathway that is conserved and downstream of the Ca2+ signal. Taken together, this study reported the novel function of FgHog1 in the regulation of Ca2+ homeostasis in F. graminearum, which advance the understanding of the HOG pathway and the association between the HOG and calcineurin pathways in fungi.

Transcription Analysis of the Acid Tolerance Mechanism of Pichia kudriavzevii NBRC1279 and NBRC1664

Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis.

Two activating phosphorylation sites of Pbs2 MAP2K in the yeast HOG pathway are differentially dephosphorylated by four PP2C phosphatases Ptc1–Ptc4

To cope with an increased external osmolarity, thebudding yeastSaccharomyces cerevisiaeactivates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.

Myxobacterial Outer Membrane β-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The β-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and β-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The β-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 μg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of β-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to β-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that β-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and β-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to β-1,6-glucanase GluM treatment (0.08 μg/mL) than β-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the β-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the β-1,3-glucan.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

The calcium-calcineurin and high-osmolarity glycerol pathways co-regulate tebuconazole sensitivity and pathogenicity in Fusarium graminearum

The calcium-calcineurin and high-osmolarity glycerol (HOG) pathways play crucial roles in fungal development, pathogenicity, and in responses to various environmental stresses. However, interaction of these pathways in regulating fungicide sensitivity remains largely unknown in phytopathogenic fungi. In this study, we investigated the function of the calcium-calcineurin signalling pathway in Fusarium graminearum, the causal agent of Fusarium head blight. Inhibitors of Ca2+ and calcineurin enhanced antifungal activity of tebuconazole (an azole fungicide) against F. graminearum. Deletion of the putative downstream transcription factor FgCrz1 resulted in significantly increased sensitivity of F. graminearum to tebuconazole. FgCrz1-GFP was translocated to the nucleus upon tebuconazole treatment in a calcineurin-dependent manner. In addition, deletion of FgCrz1 increased the phosphorylation of FgHog1 in response to tebuconazole. Moreover, the calcium-calcineurin and HOG signalling pathways exhibited synergistic effect in regulating pathogenicity and sensitivity of F. graminearum to tebuconazole and multiple other stresses. RNA-seq data revealed that FgCrz1 regulated expression of a set of non-CYP51 genes that are associated with tebuconazole sensitivity, including multidrug transporters, membrane lipid biosynthesis and metabolism, and cell wall organization. Our findings demonstrate that the calcium-calcineurin and HOG pathways act coordinately to orchestrate tebuconazole sensitivity and pathogenicity in F. graminearum, which may provide novel insights in management of Fusarium disease.

Strain-dependent differences in coordination of yeast signalling networks.

The yeast mitogen-activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and ∑1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the ∑1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose-dependent manner. In the YPH499 background, only dampening is dose-dependent. Furthermore, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the ∑1278b background only. The mitogen-activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the ∑1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the ∑1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signalling network interactions are tuned by genetic variation without significant alteration to network structure.